The principle of limited cell division potential of somatic cells in vitro is well established. In a number of fields, researchers utilize somatic cell cultures derived from normal tissues in order to study the mechanisms underlying intercellular interaction and cellular response to various stimuli. These include such diverse pursuits as evaluating the carcinogenicity of selected agents, determining the activities of various hormones, monitoring the reactions of chemotherapeutic agents, and in general studying the metabolic characteristics of a given cell type. However, the phenomenon of limited cell division of normal cells complicates these efforts and often prevents long-term evaluation of cell sensitivity and induced expression.
More specifically, the study by oncologists of neoplastic transformation of epithelial cells has been severely limited by the relatively limited in vitro population longevity. This has led to the use of human fibroblast cultures which have greater in vitro longevity as the accepted model for transformation studies. However, it has been noted that the analogy drawn between these two distinct cell-types is tenuous at best and that the lack of a true long-term epithelial model has hindered cancer research. This is despite the fact that neoplasms of epithelial origin are the most prevalent type of cancer in humans.
In particular, it is known that conventional human mammary epithelial cells generally have at most a limited cell division potential (generally about 13 doublings). In "A Simplified Method For Passage and Long-Term Growth of Human Mammary Epithelial Cells," In Vitro Cellular and Developmental Biology, Vol. 22, No. 1, January, 1986, which is incorporated herein by reference, a method of culturing non-neoplastic human mammary epithelial cells which extends the population longevity of these cells beyond the previously reported limit of 13 doublings to more than 50 generations was reported. Longevity was achieved by reducing the Ca.sup.++ concentration of the media which in turn reduced an inhibition effect in which glucocorticoids induced terminal differentiation. A significant observation which was made in these studies was that conventional human mammary epithelial cells in culture media greater than 0.06 mM ionic calcium underwent terminal differentiation after only three or four passages from primary culture.
More recently, in U.S. Pat. No. 5,026,637, filed Feb. 28, 1989, an immortal human mammary epithelial cell line, which contained sublines designated MCF-10A and MCF-10F, was described. This earlier patent application, which is assigned to the same assignee as the present application, is hereby incorporated by reference. The cell sublines MCF-10A and MCF-10F demonstrated unlimited cell division potential and produced mammary epithelial cell proteins. These cell sublines were capable of subsisting in a high-calcium media without undergoing calcium-induced cellular senescence. Both earlier cell sublines MCF-10A and MCF-10F were non-tumorigenic in athymic mice and demonstrated a characteristic response to treatment with insulin, epidermal growth factor, and cortisol.
A new immortal human mammary epithelial cell line, described herein, has been developed. This cell line (designated MCF-12) was derived from reduction mammoplasty tissue from a nulliparous postmenopausal subject. Cell line MCF-12 became immortal after exposure to elevated temperatures. Cell line MCF-12 consists of two sublines, designated MCF-12A and MCF-12F.
It is desirable to provide several genetically different, non-neoplastic epithelial cell lines which could be cultured indefinitely to permit long-term evaluation of suspected reactive agents. It would also be desirable to provide such cell lines which produce a complement of proteins characteristic of normal human breast epithelial cells. It would be further desirable to provide a method by which epithelial cell sensitivity to suspected reactive agents and cellular expression thereby induced could be studied on a long-term basis. Different interactions of the reactive agents and cellular expression in the various cell lines and sublines may prove especially useful. The present invention satisfies these goals by providing a new non-neoplastic human mammary epithelial cell line for use in cell culture studies.